Project Summary/Abstract Fc g receptors (FcgRs) trigger cell-mediated cytotoxic mechanisms following interactions with immunoglobulin G molecules clustered on the surface of a target molecule, protecting the host. Extensive post- translational modifications (PTMs) on both the FcgRs and IgG are required for this interaction, including asparagine-linked glycosylation (N-glycans). IgG N-glycans vary in response to stress and disease and compositional variability of the N-glycan can modify the affinity for FcgRIIIa (CD16a) by as much as 8-fold in vitro. FcgRs are more heavily modified than IgG with 2-7 N-glycans as well as a handful of other known modifications, and the Barb lab demonstrated that CD16a N-glycan composition impacts affinity by as much as 100-fold. Thus, FcgR N-glycans impact affinity more than the IgG N-glycans. However, almost nothing is known regarding FcgR composition within the human body, how composition changes in response to stress, or how composition impacts the structure and function of the effector cell itself (excluding preliminary descriptions of CD16a and CD16b). The successful completion of the experiments outlined in this proposal will provide unprecedented detail regarding the modification of FcgRs and how these modifications impact function of the immune system. These results will also emphasize the importance of defining PTMs from endogenous tissue for functional studies as well as provide techniques to overcome common barriers that limit studies of endogenous receptors. In Aim 1.1, we will define the composition of FcgR PTMs, including: CD16a from macrophages, CD32a from neutrophils, monocytes, macrophages, platelets and basophils; CD32b from B cells and basophils; and CD64 from monocytes and macrophages. In Aim 1.2, we will isolate NK cells and monocytes from patients with sickle cell anemia, myasthenia gravis and neuromyelitis optica to measure the cytotoxicity of these cells and define the composition of CD16a and CD64. In Aim 2, we will characterize the structure of the immune synapse and the locations of specific receptors and co-receptors, the kinetics of cell killing, as well as the kinetics of synapse formation and receptor motion using NK cells and monocytes from the patients. These results will be compared to the characterization of PTMs on the cells completed in Aim 1. Aim 2 will also study how specific FcgR PTMs impact immune cell function using cultured cells. Multiple sources of data from both Aims will be collected using samples from a single donor to identify variability in the donor population. By comparing the large amount of data collected, we expect to identify specific features of PTM composition that impact immune cell function and that represent novel therapeutic targets.